1. Field of the Invention (Technical Field)
The present invention relates to peptide, peptidomimetic, peptide-like and metallo-constructs, particularly for use in receptor-specific compositions for biological, pharmaceutical and radiopharmaceutical applications, in which the construct is conformationally fixed, with the biological-function domain generally having increased affinity for its target, upon labeling of the metal ion-binding backbone with a metal ion.
2. Background Art
Peptide Drugs. In recent years, a significant number of peptides with various biological effects have been discovered. These peptides are being explored for use as drugs, in treatment or prevention of a variety of diseases. There are significant limitations with use of peptide drugs, including extremely rapid clearance from the circulatory system, low target affinity with some peptides, immunogenicity of larger peptide constructs, and lack of stability against proteolytic enzymes. However, there are peptides in use or under investigation as therapeutic agents for a number of conditions, including somatostatin analogues, arginine vasopressin, oxytocin, luteinizing hormone releasing hormone, angiotensin-converting enzyme, renin and elastase inhibitors, as well as a variety of antagonists, including fibrinogen receptor antagonists, and the like. In addition, peptidomimetic antibiotics and peptide-based vaccines are also in use or development as human drugs.
The problems of immunogenicity and short circulatory half-life are well known, and various modifications to peptide-based drugs have been proposed in attempts to solve these problems. These include the modification of peptides or proteins with a variety of polymers, such as polyethylene glycol (PEG) and polypropylene glycol (PPG). Thus, in U.S. Pat. No. 5,091,176, Polymer-Modified Peptide Drugs Having Enhanced Biological and Pharmacological Activities, to Braatz J A and Heifetz A H, a method is set forth for making polymer-modified drugs, with reduced immunogenicity, increased circulation half-life, and enhanced potency. A different method is disclosed in U.S. Pat. No. 5,214,131, Polyethylene Glycol Derivatives, Modified Peptides and Production Thereof, to Sano A, Maeda H, Kai Y and One K.
Peptide-Based Radiopharmaceutical Drugs. Biologically active peptides, which are peptides which bind to specific cell surface receptors, have received some consideration for use as radiopharmaceuticals. Canadian Patent Application 2,016,235, Labeled Chemotactic Peptides to Image Focal Sites of Infection or Inflammation, teaches a method of detecting a site of infection or inflammation, and a method for treating such infection or inflammation, by administration of a labeled or therapeutically-conjugated chemotactic peptide. In this application, the chemotactic peptides are chemically conjugated to DTPA and subsequently labeled with .sup.111 In. The utility of DTPA chelates covalently coupled to polypeptides and similar substances is well known in the art. See, for example, U.S. Pat. Nos. 4,479,930 and 4,668,503 to Hnatowich D J. Other bifunctional chelates for radiolabeling peptides, polypeptides and proteins are well known in the art. Biologically active peptides are described in U.S. Pat. No. 4,427,646, Use of Radiolabeled Peptide Derived From Crosslinked Fibrin to Locate Thrombi In Vivo, to Olexa S A, Knight L C and Budzynski A Z, in which iodination is discussed as a means of radiolabeling. In U.S. Pat. No. 5,371,184, Radiolabelled Peptide Compounds, to Rajagopalan R, Lyle L R and Dunn T J, hirudin receptor-specific peptides, radiolabeled via a chelate ligand, are disclosed. In U.S. Pat. No. 4,986,979, Imaging Tissue Sites of Inflammation, to Morgan C A Jr and Anderson D C, use of chelates and direct iodination is disclosed. In U.S. Pat. No. 4,732,864, Trace-Labeled Conjugates of Metallothionein and Target-Seeking Biologically Active Molecules, to Tolman G L, the use of metallothionein or metallothionein fragments conjugated to a biologically active molecule, including peptides, is disclosed. In Dean R T and Lister-James J, International Application No. PCT/US93/05372, Technetium-99m Labeled Peptides for Imaging; Dean R T and Lister-James J, International Application No. PCT/US93/04794, Technetium-99m Labeled Peptides for Thrombus Imaging; Dean R T, Buttram S, McBride W, Lister-James J, and Civitello E R, International Application No. PCT/US93/03687, Technetium-99m Labeled Peptides for Imaging; Dean R T, Lees R S, Buttram S and Lister-James J, International Application No. PCT/US93/02320, Technetium-99m Labeled Peptides for Imaging Inflammation; and Dean R T, McBride W and Buttram S, International Application No. PCT/US92/10716, Technetium-99m Labeled Peptides for Imaging a variety of peptide constructs are disclosed, all involving a Tc-99m binding moiety covalently or otherwise linked to the peptide, or to a polyvalent linker moiety, which is itself linked to one or more peptides. These previous methods all employ some conjugation means with a chelator in order to effectuate labeling with a radionuclide or other medically useful metal ion, such as a paramagnetic contrast agent. The only exception involves direct radioiodination; the iodine labeling of proteins or peptides containing tyrosine or histidine residues is well known, for example, by the chloramine-T, iodine monochloride, Iodogen or lactoperoxidase methods.
In U.S. Pat. No. 5,225,180, Technetium-99m Labeled Somatostatin-Derived Peptides for Imaging, to Dean R T, Lister-James J and Buttram S, technetium-99m labeling of peptides containing at least two cysteine residues capable of forming a disulfide bond through reduction of the disulfide is disclosed. Other somatostatin-based radiopharmaceuticals are disclosed in U.S. Pat. No. 5,382,654, Radiolabelled Peptide Compounds, to Lyle L R, Rajagopalan R, and Deutsch K; Albert R and Macke H, European Patent Application No. EP948 10008.6, Somatostatin Analogs Containing Chelating Groups and Their Radiolabeled Compositions; Dean R T, McBride W and Lister-James J, International Application No. PCT/US94/06274, Radiolabeled Somatostatin-Derived Peptides for Imaging and Therapeutic Uses; and McBride W and Dean R T, International Application No. PCT/US94/08335, Somatostatin Derivatives and Their Radiolabelled Products. Use of peptide radiopharmaceuticals in general, not limited to somatostatin analogues, and various examples thereof, are given in Fischman A J, Babich J W, Strauss H W: A Ticket to Ride: Peptide Radiopharmaceuticals. J Nucl Med 34:2253-2263, 1993. A method of metal chelation, using amino acid sequences that are capable of forming metal complexes and which are directly incorporated into peptides at nonbiologically active locations has been disclosed. U.S. Pat. No. 5,464,934, Metal Chelates as Spacer Compounds in Biologically Active Peptides, to Dunn T J, Srivivasan A, Lyle L R, Rajagpalan R.
Other biologically active peptides include analogues of formyl peptide chemoattractants which bind to neutrophils. These peptides are based on the sequence N-formyl-Met-Leu-Phe. The clinical and diagnostic imaging potential of formylated chemotactic peptides has been demonstrated by Fischman et al. (Fischman A J, Pike M C, Kroon D, Fucello A J, Rexinger D, tenKate C, Wilkinson R, Rubin R H and Strauss H W: Imaging focal sites of bacterial infection in rats with indium-111-labeled chemotactic peptide analogs. J Nucl Med 32:483-491, 1991) using chemotactic peptides chemically conjugated to DTPA and subsequently labeled with .sup.111 In. Chemotactic peptides have also been radioiodinated by synthesizing formylated peptides containing tyrosine amino acids. These peptides have been used in vitro and have the same biological function as unlabeled formylated peptides (Janeczek A H, Marasco W A, Van Alten P J and Walter R B: Autoradiographic analysis of formylpeptide chemoattractant binding, uptake and intracellular processing by neutrophils. J Cell Sci 94:155-168, 1989). Finally, chemotactic peptides have also been labeled with .sup.99m Tc using a nicotinyl hydrazine bifunctional chelate approach (Babich J W, Graham W, Barrow S A, Dragotakes S C, Tompkins R G, Rubin R H and Fischman A J: Technetium-99m-labeled chemotactic peptides: comparison with Indium-111-labeled white blood cells for localizing acute bacterial infection in the rabbit. J Nucl Med 34:2176-2181, 1993).
Peptides containing the adhesive sequence RGD are under active investigation as anti-thrombotic agents (Imura Y, Stassen J-M, Dunting S, Stockmans F, and Collen D: Antithrombotic properties of L-cysteine, N-(mercaptoacetyl)-D-Tyr-Arg-Gly-Asp-sulfoxide (G4120) in hamster platelet-rich femoral vein thrombosis model, Blood 80:1247-1253, 1992). Knight et al. (Knight L C, Radcliffe R, Maurer A H, Rodwell J D and Alvarez V L: Thrombus imaging with Tc-99m synthetic peptides based upon the binding domain of a monoclonal antibody to activated platelets. J Nucl Med 35:282-288, 1994) have reported on the use of .sup.99m Tc-synthetic peptide-metallothionein complexes, containing the radiometal binding sequence Lys-Cys-Thr-Cys-Cys-Ala, which bind to the platelet glycoprotein IIb/IIIa complex to image fresh thrombi in jugular and femoral veins. Other RGD-containing sequences are disclosed in U.S. Pat. No. 5,395,609, Synthetic Peptides for Use in Tumor Detection, to Stuttle A W J.
Radiolabeled peptide constructs, with two binding sequences coupled to DTPA, have been reported. A dimer .sup.111 In-DTPA-labeled laminin sequence was prepared for tumor imaging, in which the dimer was formed by reacting a peptide sequence containing a single YIGSR with DTPA dianhydride, yielding a dimer represented by the formula DTPA-(GYIGSR-NH.sub.2).sub.2. In preliminary studies the dimer was more potent than a peptide with a single YIGSR sequence. Swanson D, Epperly M, Brown M L et al: In-111 laminin peptide fragments for malignant tumor detection. J Nucl Med 34:23 1P, 1993 (Abstract). A dimer of a melanotropin analogue linked to .sup.111 In-DTPA in a similar fashion has also been reported as an imaging agent for metastatic melanoma. Wraight E P, Bard D R, Maughan T S et al, Br J Radiology 65:112-118, 1992; and Bard D R, Wraight E P, Knight C G: BisMSH-DTPA: a potential imaging agent for malignant melanoma. Ann NY Acad Sci 680:451-453, 1993.
Structure of Peptides. The folding of linear chain amino acids in peptides and proteins in a very distinctive manner is responsible for their unique three dimensional structure. It is now clear that the side chains of individual amino acids have a preferential propensity to nucleate a particular secondary structure (Chou P Y and Fasman G D: Prediction of the secondary structure of proteins from their amino acid sequence. In Advances in Enzymology, Vol. 47 (1978) pp. 45-145, John Wiley & Sons, New York). The properties of these side chains, such as steric bulk and inherent hydropathicity, cause the peptide chain to fold as a helix, sheet, or a reversed turn. In addition to these local effects, both covalent as well as noncovalent interactions between distant as well as adjacent amino acids in the chain also play a very important role in determining, stabilizing and biasing a particular three dimensional structure. Examples of noncovalent interactions include hydrophobic interactions, van der Waals' forces, and hydrogen bonds. Electrostatic interactions in the form of a salt bridge between a positively charged side chain and a negatively charged side chain are common, and stabilize a peptide or protein in a particular configuration. The most important type of covalent interaction between two amino acids in a chain is the formation of a disulfide linkage between two Cys residues that nucleates a particular conformational preference in the molecules. These interactions can be short range (local or regional) or long range (global).
Most of the elements for inducing and stabilizing a conformational preference in naturally occurring proteins and peptides have been used to design and synthesize a wide variety of peptide analogues with preferred or biased conformational characteristics. Examples of structural changes in peptides to cause conformational bias and restriction have been discussed in the literature (Hruby V J: Conformational restrictions of biologically active peptides via amino acid side chain groups. Life Sciences 31:189-199, 1981). The incorporation of modified amino acids, such as N.sup.a -Methyl or C.sup.a -Methyl amino acids or other designer amino acids with conformationally restricted side chains, causes a strong local conformational effect. In synthetic peptides long range or global conformational restriction can routinely be achieved by cyclizing a peptide through appropriate amino acid end groups or side chains. The types of cyclic bridges commonly employed are disulfide bridges between two Cys residues in the peptide chain, and related thioester and thioether bridges, and formation of a lactam or lactone bridge between appropriate chemical groups in the amino acid side chains. Numerous highly potent analogues of many biologically active peptides have been designed using these approaches. Examples include peptide hormones such as somatostatin, opioid peptide, melanotropin, neurokinins, glucagon, and ACTH analogues. Hruby V J, Sharma S D, Collins N, Matsunaga T O and Russel K C: Applications of synthetic peptides, in Synthetic Peptides, A User's Guide, Grant G A, editor, W. H Freedman and Company, 1992, pp. 259-345.
Peptide-Metal Ion Interaction. Metal ion complexation within a given amino acid sequence, such as encountered in certain proteins, also appears to effect conformational restriction. Specific structures, called Zinc fingers, in various DNA transcription factors result from complexation of Zn ions to a specific amino acid sequence in the protein. In Vallee B L and Auld D S: Zinc coordination, function, and structure of zinc enzymes and other proteins, Biochemistry 29:5648-5659, 1990, the general characteristics of non-metallothionein proteins which contain zinc binding sites are described. Similarly, a family of calcium binding proteins, including calmodulin and related proteins, have highly conserved domains for complexation of Ca ions. These metal binding proteins have unique functional roles in the body that are displayed after the metal ion has complexed to them. The complexation process is known to cause a switch in conformational characteristics which in turn triggers the functional response exerted by the protein.
The area of peptide-metal ion complexation receiving the most interest involves zinc fingers, natural sequences with specific Zn binding domains in transcription proteins that mediate gene regulation (Rhodes D and Klug A: Zinc fingers. Scientific American 268(2):56-65, 1993). The reported zinc fingers which have been synthesized and studied for metal binding characteristics in respect to confornational restriction and peptide folding are not of biological relevance, since they are not capable of establishing site-specific interactions with DNA in a manner similar to the transcription proteins that incorporate these zinc fingers. Krizek B A, Amann B T, Kilfoil V J, Merkle D L, and Berg J M: A consensus zinc finger peptide: Design, high affinity metal binding, a pH-dependent structure, and a His to Cys sequence variant. J Amer Chem Soc 113:4518-4523, 1991.
Metal ion induced switches in the tertiary structure of synthetic peptides have been shown in some model studies. Reid, Hodges and co-workers (Shaw G S, Hodges R S, Sykes B D: Calcium-induced peptide association to form an intact protein domain: 1H NMR structural evidence. Science 249:280, 1990; and Reid R E, Gariepy J, Saund A K, Hodges R S: J Biol Chem. 256:2742, 1981) showed that a peptide fragment related to a natural calcium binding protein exhibits enhanced .alpha.-helical structure upon binding to calcium. This is due to dimerization of two helical peptide segments located at each end, which is induced by complexation of a calcium ion in the middle peptide segment. Sasaki and co-workers (Lieberman M, Sasaki T: J Am Chem Soc 113:1470, 1991) have attached a metal binding chelator to one end of a peptide with a low propensity to form an .alpha.-helical structure. Upon complexation with an iron ion three peptide-chelator molecules complex with one metal ion to form a helix bundle. Formation of three-dimensional arrays of the existing secondary structure in these examples, although caused by the complexing metal ion, is not entirely stabilized by it. The helical segments involved in forming a bundle of two or three helices are amphiphilic. The main role of the complexing metal ions in these cases has been to bring these amphiphilic helices close enough so that they interact with each other through amphiphilic interactions, thereby stabilizing the helical bundle.
Stabilization of the alpha helix in short peptides has been reported by making an exchange-inert ruthenium.sup.III complex (Ghadiri M R and Femholz A K: Peptide architecture. Design of stable .alpha.-helical metallopeptides via a novel exchange-inert Ru.sup.III complex. J Am Chem Soc 112:9633-9635, 1990) or exchange-labile Cu, Zn, or Cd complex (Ghadiri M R and Choi C: Secondary structure nucleation in peptides. Transition metal ion stabilized .alpha.-helices. J Am Chem Soc 112:1630-1632, 1990) with peptides that have a propensity to form helical structures. In these 17 amino acid-long peptides two His residues or a Cys and a His residue were placed at i and i+4 positions which would reside on the same side of two consecutive turns in an .alpha.-helix and formed an exchange-inert complex with cis[Ru(III)(NH.sub.3).sub.4 (H.sub.2 O).sub.2 ].sup.2+ or exchange-labile complex with Zn, Cu, or Cd. The resulting complexes were shown by circular dichroism studies to be of higher helical content. In this art, incorporated generally into U.S. Pat. No. 5,200,504, Metallopeptides Having Stabilized Secondary Structures, to Ghadiri M R; U.S. Pat. No. 5,408,036, Isolated Metallopeptide: Compositions and Synthetic Methods, to Ghadiri M R; U.S. Pat. No. 5,410,020, Methods for Preparing Metallopeptides Having Stabilized Secondary Structures, to Ghadiri M R, the peptide molecule provides only two of the metal chelation sites. The other valences of the metal coordination sphere are satisfied by other unidentate ligands such as NH.sub.3. H.sub.2 O, solvents or halide atoms. Another distinguishing feature of this art is that the two metal complexation sites in the peptide are provided by distant (non-contiguous) amino acids separated by at least two or more amino acids. This method has also been used to induce metal ion-assisted spontaneous self-assembly of polypeptides into three helix (Ghadiri M R, Soares C, Choi C: A convergent approach to protein design. Metal ion-assisted spontaneous self-assembly of a polypeptide into a triple-helix bundle protein. J Am Chem Soc 114:825-831, 1992) and four-helix bundles (Ghadiri M R, Soares C, Choi C: Design of an artificial four-helix bundle metalloprotein via novel Ruthenium(II)-assisted self-assembly process. J Am Chem Soc 114:4000-4002, 1992). In both cases, an amphiphilic polypeptide designed with the propensity to form an .alpha.-helix, with a metal chelator attached at its N-terminus, was complexed to a metal ion which caused it to trimerize or tetramerize with very high helical content. It is evident that the resulting helical bundle was composed of homomeric chains. Formation of metal ion assisted helical bundles with heteromeric polypeptide chains has not yet been demonstrated.
Peptide Libraries and Combinatorial Chemistry. Combinatorial chemistry techniques are now well recognized tools for rapid drug discovery. A library of peptides and other small molecules, with its enormous pool of structurally diverse molecules, is well suited for both lead generation as well as lead optimization. Libraries of a variety of molecular species have been described in literature and screened for drug discovery. These molecular species include peptides, peptoids, peptidomimetics, oligonucleotides, benzodiazepines, and other libraries of small organic molecules.
Various approaches used to construct a library of structurally diverse chemical compounds include chemical synthesis and genetic engineering methods. Chemically synthesized libraries can be either soluble (a mixture of various compounds in a solution) or solid (compounds synthesized on a solid surface). Libraries produced by genetic engineering tools are largely composed of peptide molecules, and are similar to solid-phase libraries in the sense that the peptide molecules are displayed or attached on the surface of vectors or bacteriophages used for their production.
The prior art on designing, synthesizing, screening, and evaluation of peptide-based libraries has been reviewed in the following articles, incorporated herein by reference: Pinilla C et al: A review of the utility of soluble peptide combinatorial libraries. Biopolymers (Peptide Sci) 37:221-240, 1995; Lebl M et al: One-bead-one-structure combinatorial libraries. Biopolymers(Peptide Sci) 37:177-198, 1995; Holmes C P et al: The use of light-directed combinatorial peptide synthesis in epitope mapping. Biopolymers(Peptide Sci) 37:199-211, 1995; and, Moran E J et al: Novel biopolymers for drug discovery. Biopolymers(Peptide Sci) 37:213-219, 1995.
The prior art in construction and screening of small molecule libraries, including non-peptide libraries, has recently been reviewed extensively in a "Special Issue on Combinatorial Libraries" appearing in Accounts of Chemical Sciences, 29:111-170, 1996. Articles therein applicable hereto include: Czarnik A W: Guest Editorial, at 112-113; DeWitt S H et al: Combinatorial organic synthesis using Parke-Davis's DIVERSOMER method, at 114-122; Armstrong R W et al: Multiple-component condensation strategies for combinatorial library synthesis, at 123-131; ElIman J A: Design, synthesis, and evaluation of small molecule libraries, at 132-143; Gordon E M et al: Strategy and tactics in combinatorial organic synthesis. Applications to drug discovery, at 144-154; Still WC: Discovery of sequence selective peptide binding by synthetic receptors using encoded combinatorial libraries, at 155-163; and, Hsieh-Wilson L C et al: Lessons from the immune system: From catalysis to material sciences, at 164-170. Also of note is Thompson L A and Ellman J A: Synthesis and applications of small molecule libraries. Chem Rev 96:555-600, 1996. The teachings of all the foregoing articles are incorporated by reference.
Phage Display Libraries. Phage display methods of preparing large libraries of peptides (up to 10.sup.6 -10.sup.8 chemically different peptides) are now well established (Scott and Smith: Science 249:386-390, 1990; Devlin et al: Science 249:404-406, 1990; Cwirala et al: Proc Natl Acad Sci USA 87:6378-6382, 1990; and U.S. Pat. Nos. 5,432,018; 5,338,665; and 5,270,170). In these libraries, the individual peptides are displayed on the surface of bacteriophages or other suitable vectors and are used in screening assays against the target receptor. Because of inherent properties of biological systems, these methods in general are limited to construction of simple straight-chain peptide libraries with only natural amino acids. These methods also do not allow for further chemical modification in the peptides after the construction of a phage display library.
Spatially Addressable Parallel Synthesis of Solid Phase Bound Libraries. Various strategies for chemical construction of a library of peptides or other small molecules are also well established. One strategy involves spatially separate synthesis of compounds in parallel on solid phase or on a solid surface in a predetermined fashion so that the location of one compound or a subset of compounds on the solid surface is known. The first such method was developed by Geysen for peptide epitope mapping (Geysen H M, Meloen R H, Barteling S J: Proc Natl Acad Sci USA 81:3998-4002, 1984). This method involves synthesis of various sets and subsets of a library of peptides on a multiple number of polypropylene pin tips in a predetermined fashion. The screening of these pin-based peptides is done by immersing one pin per well, the well containing the assay reagents and components, in multiwell titer plates. Pin loading levels range from 100 nM to 50 .mu.M, which is sufficient for conducting multiple biological assays. The assembly of a library of greater than 10,000 molecules by this method is, however, cumbersome and time consuming. The "light-directed spatially addressable parallel chemical synthesis" technique (Fodor SPA et al: Science 251:767-773, 1991), based upon use of photolithographic techniques in peptide synthesis on a solid surface, such as a borosilicate glass microscope slide, is a better method of constructing libraries containing more than 100,000 spatially separated compounds in a pre-determined fashion. However, synthesis of libraries that are structurally more diverse than simple peptides requires the development of orthogonal photolabile protecting groups that can be cleaved at different wavelengths of light. In addition, the solid surface bearing these libraries also has been reported to cause a pronounced effect on binding affinities in library screening assays (Cho CY et al: Science 261:1303-1305, 1993; Holmes C P et al: Biopolymers 37:199-211, 1995).
The DIVERSOMER.RTM. apparatus designed by DeWitt and coworkers at Parke-Davis Pharmaceutical Research Division of Warner-Lambert Company, Ann Arbor, Mich., USA, offers a convenient and automated parallel synthesis of small organic molecule libraries on a solid phase (DeWitt S H et al: Proc Natl Acad Sci USA 90:6909-6913, 1993; U.S. Pat. No. 5,324,483; DeWitt S H et al: Acc Chem Res 29:114-122, 1996). Another conceptually similar apparatus for the solid phase synthesis of small organic molecule libraries has been reported by Meyers and coworkers (Meyers H V et al: Molecular Diversity 1:13-20, 1995). A commercial instrument is also now available (Advanced ChemTech Inc, Louisville, Ky., USA). This instrument can produce 96 different compounds in a parallel synthesis and is compatible with a wide range of reaction conditions, temperatures, mix times and other parameters.
Pooling and Split Synthesis Strategies. Large libraries of compounds are assembled by a pooling strategy that employs equimolar mixtures of reactants in each synthetic step (Geysen H M et al: Mol Immunol 23:709-715, 1986) or preferably by adjusting the relative concentration of various reactants in the mixture according to their reactivities in each of the coupling reactions (Ostresh J M et al: Biopolymers 34:1681-1689, 1994; U.S. Pat. No. 5,010,175 to Rytter W J and Santi D V). The split synthesis approach was pioneered by A. Furka (Furka A et al, (1988), 14th International Congress of Biochemistry, Vol. 5, Abstract No. FR:013; Furka A et al: Int J Peptide Protein Res 37:487-493, 1991; Sebestyen F et al: BioMed Chem Lett 3:413-418, 1993), in which equimolar mixtures of compounds are obtained by splitting the resin in equal portions, each of which is separately reacted with each of the various monomeric reagents. The resin is mixed, processed for the next coupling, and again split into equal portions for separate reaction with individual reagents. The process is repeated as required to obtain a library of desired oligomeric length and size. This approach is also the basis of the "one-bead one-peptide" strategy of Lam et al. (Lam K S et al: Nature 354:82-84, 1991; Lam K S et al: Nature 360:768, 1992) which employs amino acid sequencing to ascertain the primary structure of the peptide on a hit bead in a bioassay. Automated systems have been developed for carrying out split synthesis of these libraries with rather more efficiency (Zukermann R N et al: Peptide Res 5:169-174, 1992; Zukermann R N et al: Int J Peptide Protein Res 40:497-506, 1992). A common artifact occasionally seen with all these resin bound libraries is altered target-specific affinity by some solid phase bound compounds in bioassays, which can result in totally misleading results. Another highly successful strategy that overcomes this problem is construction of soluble libraries (Houghten R A et al: Proc Natl Acad Sci USA 82:5131-5135, 1985; Berg et al: J Am Chem Soc 111:8024-8026, 1989; Dooley C T et al: Science 266:2019-2022, 1994; Blondelle S E: Antimicrob Agents Chemother 38:2280-2286, 1994; Panilla C: Biopolymers 37:221-240, 1995). This strategy involves a deconvolution process of iterative re-synthesis and bioassaying until all the initially randomized amino acid positions are defined. Several modifications to this strategy have also been suggested. For example, co-synthesis of two libraries containing orthogonal pools, as demonstrated by Tartar and coworkers, eliminates the need of iterative re-synthesis and evaluation (Deprez B et al: J Am Chem Soc 117: 5405-5406, 1995). The positional scanning method devised by Houghton and coworkers eliminates iterative re-synthesis (Dooley C T et al: Life Sci 52:1509-1517, 1993; Pinilla C et al: Biotechniques 13:901-905, 1992; Pinilla C et al: Drug Dev Res 33:133-145, 1992). A combination of this strategy with the split synthesis methods described above has also been proposed (Erb E et al: Proc Natl Acad Sci USA, 91:11422-11426, 1994). A major problem with the soluble library approach involves its successful applicability to high affinity systems. The abundance of each compound in solution can be influenced by the total number of compounds in a library which can affect the biological activity. For this reason, a highly active compound in any pool may not in fact be the most potent molecule. Lack of reasonable solubilities of certain members in a library may further influence this phenomenon. In fact, for several libraries the most active peptide was not even identified in the most active library pool (Dooley C T et al: Life Sci 52:1509-1517, 1993; Eichler J, in Proc. 23rd Eur. Peptide Symp., Berga, September 1994, Poster 198; Wyatt J R: Proc Natl Acad Sci USA, 91:1356-1360, 1994).
Various strategies for determination of the structure for a positive hit in a random library have been developed. For a solid-phase library, direct analytical modalities include Edman degradation for peptide libraries, DNA sequencing of oligonucleotide libraries, and various mass spectrometry techniques on matrix bound compounds. The technique of creating a series of partially end-capped compounds at each of the synthetic steps during library assembly helps their unambiguous identification by mass spectrometry (Youngquist R S et al: J Am Chem Soc 117:3900-3906, 1995; Youngquist R S et al: Rapid Commun Mass Spectr 8:77-81, 1994). This technique has been claimed to be universally applicable to a wide variety of chemically diverse libraries. Direct mass spectrometric analysis of compounds covalently bound to a solid phase matrix of particles is also now possible by the use of matrix-assisted laser desorption/ionization (MALDI) techniques (Siuzadak G et al: Bioorg Med Chem Lett 6:979, 1996; Brown B B et al: Molecular Diversity 1:4-12, 1995). In addition to these analytical techniques, various encoding strategies have been devised for structure elucidation in organic molecule-based libraries, including both non-peptide and non-nucleotide libraries. Various coding strategies include DNA encoding, peptide coding, haloaromatic tag encoding, and encoding based on radiofrequency transponders.
Most of the libraries described above are termed "random" libraries because of their enormous structural and conformational diversity. Libraries of relatively restricted and biased structures have also been reported. Examples of libraries of conformationally rigid compounds built on a structurally common template include benzodiazepine, .beta.-lactam, .beta.-turn mimetics, diketopiperazines, isoquinolines, dihydro- and tetrahydroisoquinolines, 1,4 dihydropyridines, hydantoins, pyrrolidines, thiazolidine4-carboxylic acids, 4-thiazolidines and related 4-metathiazanones and imidazoles.
Among the various classes of libraries of small molecules, peptide libraries remain the most versatile because of the structural diversity offered by the use of naturally occurring amino acids, incorporation of a variety of designer amino acids, and the high efficiency and ease with which peptide synthesis can be accomplished. In addition, another level of structural diversity in peptide-based libraries has been added by post-synthesis modification of the libraries. These modifications include permethylation, acylation, functionalization of the side chain functionality, and reductive amination of the N-terminus.
Many libraries specifically customized for one particular biological target have also been reported. These libraries are generally assembled by incorporating only a set of structural elements that might be essential for eliciting a target-specific response. Some of the reported libraries of this class include aspartic acid protease, zinc proteases, carbonic anhydrase inhibitors, tyrosine kinase inhibitors, estrogen receptor ligands, and antioxidants.